The need to increase the rooting response of plant cultures is important when trying to produce identical copies of a mature plant. Mature plant cultures are generally difficult to propagate. However, it is necessary to use mature plant tissue to determine the characteristics or properties of progeny tissues. For example, it is not possible to determine whether a culture is blight resistant unless the culture is a mature plant. If the mature plant is resistant, then copies of the plant will also be resistant. Thus, it is desirable to have a method for growing tissue cultures from mature plants.
Scientists and industry have had a difficult time culturing certain types of plant tissue. Mature tissue samples do not develop into functioning plants when grown in a clear medium. The leaves are not formed properly, and the roots do not form properly, if at all. This difficulty may be due to the fact that the upper portion of a plant culture requires light while the lower portion requires darkness. In addition, certain substances which are required for growth and development of plant cultures are most effective in dark conditions. When these substances are exposed to light, which is often the case in conventional, in vitro, rooting systems, they break down. The break down of these substances prevents proper culture development.
Attempts have been made to create an in vitro environment that offered darkness to the lower portion of the culture and light to the upper portion of a culture. One prior conventional rooting system utilized charcoal in the rooting medium. Charcoal is added directly to the lower medium in an amount rendering the entire medium opaque. This represents an effective method for blocking light from the culture while allowing the upper or exposed portion of the explant to absorb light. However, several drawbacks exist in this type of system. For instance, charcoal in even minimal concentrations has been found to absorb growth regulators or auxins, such as Indole-3-Acetic Acid (IAA) and Indole-3-Butyric Acid (IBA). These auxins are essential for tissue growth as and development. In addition to absorbing these auxins, charcoal has been shown to absorb thiamine, nicotinic acid, pyridoxine, folic acid, iron chelate and zinc. Yet another problem experienced in the use of charcoal results from the lack of consistency of such systems due to the difficulty in regulating the amount of auxins absorbed by the charcoal. This problem makes it difficult to develop standard protocols.
Another problem encountered in the use of the conventional charcoal-bearing system is that the charcoal renders observation of the growth and development of tissue difficult or impossible. To ensure the best success rate for the cultures, it is important to remove them from the system as soon as the rooting system has matured sufficiently for transfer to a soil system. The charcoal saturated medium does not allow for such inspection, thus leaving to conjecture the timing for removal of the tissue from the system.
Another known rooting system that has been employed, specifically with the American Chestnut of the genus Castanea, involves a three-medium system. The system is used in the rooting of the American chestnut due to the traditionally low success rate experienced with other conventional methods. The three-medium system involves placing an explant in a first medium for 4-8 weeks, followed by placement in a second medium for 2 weeks, and finally placement in a third medium for 3 weeks. Unfortunately, this system does not have the best success rate. In addition, this system is time consuming, rendering the system impractical for commercial applications. Moreover, each transfer of the explant increases the chances that it will become contaminated.